PMID:2651435: Difference between revisions
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[[Category:]][http://gowiki.tamu.edu/wiki/index.php/Category: GO: | [[Category:GO:0016020_!_membrane]][http://gowiki.tamu.edu/wiki/index.php/Category:GO:0016020_!_membrane GO:0016020] | ||
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When membranes are prepared using high ionic strength buffers, the KDO transferase activity is equally distributed in the 105,000 x g pellet and supernatant fractions. Using the method of Munson et al. (PMID:342527), all of the activity fractionates with membranes (pg. 6959). In subsequent work (PMID:1577828) the WaaA transferase was purified from membranes obtained after disruption of cells by passage through a French pressure cell. | When membranes are prepared using high ionic strength buffers, the KDO transferase activity is equally distributed in the 105,000 x g pellet and supernatant fractions. Using the method of Munson et al. (PMID:342527), all of the activity fractionates with membranes (pg. 6959). In subsequent work (PMID:1577828) the WaaA transferase was purified from membranes obtained after disruption of cells by passage through a French pressure cell. | ||
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Revision as of 16:43, 10 November 2008
Brozek, KA, Hosaka, K, Robertson, AD and Raetz, CR (1989) Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A.J. Biol. Chem. 264:6956-66
| Abstract | Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs. |
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Annotations
| Gene product | Qualifier | GO ID | GO term name | Evidence Code | with/from | Aspect | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
WaaA |
GO:0016740 |
transferase activity |
IDA: Inferred from Direct Assay |
F |
This enzyme sequentially transfers two residues of KDO (3-deoxy-D-manno-octulosonic acid) from CMP-KDO to tetraacyldisaccharide 1,4’ bisphosphate (lipid IVA) to form KDO2-lipid IVA. It is assayed using [4’-32P]lipid IVA and CMP-KDO. The former substrate was prepared and partially purified by the investigators while the latter is generated in situ (using CMP-Kdo synthase, Kdo and CTP) since it is extremely unstable. Product was separated from reactants by thin layer chromatography and quantified by scintillation counting (pg. 6957; see also, Figs. 5, 7, and 9). |
complete | ||
|
WaaA |
GO:0016020 |
membrane |
IDA: Inferred from Direct Assay |
C |
When membranes are prepared using high ionic strength buffers, the KDO transferase activity is equally distributed in the 105,000 x g pellet and supernatant fractions. Using the method of Munson et al. (PMID:342527[1]), all of the activity fractionates with membranes (pg. 6959). In subsequent work (PMID:1577828[2]) the WaaA transferase was purified from membranes obtained after disruption of cells by passage through a French pressure cell. |
complete | ||
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- ↑ Munson, RS Jr et al. (1978) Biosynthesis of lipid A. Enzymatic incorporation of 3-deoxy-D-mannooctulosonate into a precursor of lipid A in Salmonella typhimurium. J Biol Chem 253 1503-11 PubMed
- ↑ Belunis, CJ & Raetz, CR (1992) Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli. J. Biol. Chem. 267 9988-97 PubMed